Proteins have become commercially important as drugs that are also generally called “biologicals”. One of the greatest challenges is the development of cost effective and efficient processes for purification of proteins on a commercial scale. While many methods are now available for large-scale preparation of proteins, crude products, such as body fluids or cell harvests, contain not only the desired product but also impurities, which are difficult to separate from the desired product. Moreover, biological sources of proteins usually contain complex mixtures of materials.
Biological sources such as cell culture conditioned media from cells expressing a desired protein product may contain less impurities, in particular if the cells are grown in serum-free medium. However, the health authorities request high standards of purity for proteins intended for human administration. In addition, many purification methods may contain steps requiring application of low or high pH, high salt concentrations or other extreme conditions that may jeopardize the biological activity of a given protein. Thus, for any protein it is a challenge to establish an efficient purification process allowing for sufficient purity while retaining the biological activity of the protein.
Protein purification generally comprises at least three phases or steps, namely a capture step, in which the desired protein is separated from other components present in the fluid such as DNA or RNA, ideally also resulting in a preliminary purification, an intermediate step, in which proteins are isolated from contaminants similar in size and/or physical/chemical properties, and finally a polishing step resulting in the high level of purity that is e.g. required from proteins intended for therapeutic administration in human or animals.
Typically, the protein purification steps are based on chromatographic separation of the compounds present in a given fluid. Widely applied chromatographic methods are e.g. gel filtration, ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography or reverse-phase chromatography.
Aqueous Two-Phase Systems (A2PS) are an alternative to classical chromatographic processes. A2PS processes, have been used in the prior art for the purification of proteins (see Table 1).
TABLE 1Proteins purified in A2PSMWYieldPurificationProteinSource(kDa)PI(%)factorReferencesXylanaseBacillus395.284157(Duarte et al., 2003)pumilusβ-glucosidaseAspergillus914.69852(Johansson &nigerReczey, 1998)LipaseAcinetobacter356.296841(Bompensieri etcalcoaceticusal., 1998)Glucoamylase—524.68963(Minami & Kilikian, 1997)BSABovin105.8595—(Bleier et al., 2001)CutinaseSacharomyces246.82955(Costa et al., 2000)cervisiaeGFPEscherishia275.67913.3(Li & Beitle, 2002)coliLysozymeChicken egg28.00952.2(Balasubramaniamwhiteet al., 2003)
The purification of interferon-beta (IFN-β) from a production medium containing a partially purified serum resulted in a 350-fold purified and up to 10-fold concentrated IFN-β sample with a specific activity of 3-7.10E106 IU/mg (Menge et al., 1987). However, the application of A2PS for the purification of therapeutic proteins in industrial scale is still limited.
A2PS is based on the partitioning of the target molecule between two immiscible aqueous phases (for instance a PEG/salt system). This system is adapted for proteins extraction because the high water content of both phases (70-80% w/w) means high biocompatibility and low interfacial tension minimizing degradation of the protein. The principle of protein purification by A2PS is e.g. exemplified for the purification of the insulin-like growth factor IGF-1 in U.S. Pat. No. 5,695,958.
The choice of a suitable phase system (polymer/polymer; polymer/salt . . . ) is the key step in a purification process based on A2PS. This system must show a high selective distribution of the target protein between the phases. Examples of couples used for A2PS purification of proteins from various biological sources are illustrated in Table 2.
The composition of aqueous polymer two-phase systems is usually represented in the rectangular form of a phase diagram, as illustrated in FIG. 5, taken from Hatti-Kaul, 2000. The vertical axis is commonly used for the component, which is enriched in the top phase. Amounts of polymer/salt X, polymer/salt Y and S of water are mixed. The total composition of one mixture is represented by one of the points A1, A2, or A3 on the phase diagram. The mixtures separate into two phases. The compositions of these two phases are represented by the points T and B, which are called nodes and are located at the bimodal line. The bimodal curve is the line separating two domains of compositions: one where the system is mono-physic (left and bottom of the curve) and one where phase separation can be observed (top and right of the curve). The line joining the points B and T representing the compositions of the coexisting phases is called a tie line. The points A1, A2, or A3, representing the total mixture must be positioned on the same tie line as the node B and T characterizing the compositions of the coexisting phases originated from these mixtures.
As shown in FIG. 5, mixtures of different total compositions represented by different points on the same tie line give rise to two-phase systems with identical compositions but different volumes of the coexisting phases. The volume ratio of the two phases can be approximated graphically by the ratio of the segment AB (top phase) and AT (bottom phase).
TABLE 2Examples of protein extraction based on A2PSReferenceEnzyme or proteinSystem usedOrigin(Menge et al., 1984)InterferonPEG-dextranraw materialDE 2943026(Schütte et al., 1984)D-Lactate dehydrogenasePEG-phosphateLactobacilluscellubiosus(Kim et al., 1985)Protease, amylasePEG-dextranAspergillus orizaeU.S. Pat. No. 4,508,825(Schütte et al., 1985)L-Leucin dehydrogenasePEG-phosphateBacillus cereus(Gustafsson et al., 1986)ADH, hexokinasePEG-phosphateYeastU.S. Pat. No. 4,579,661(Gustafsson et al., 1986)TransferrinPEG-phosphateBood plasma(Paul et al., 1986)Dextran-sucrasePEG-dextranLeuconostocU.S. Pat. No. 4,591,563mesenteroides(van Wijnendaele et al., 1991)Hepatitis B antigenPEG-ammonium sulfateYeastEP 0199698(van Wijnendaele et al.,Alpha-1-antitrypsinPEG-ammonium sulfateYeast1991) EP 0199698(Dove & Mitra, 1988)Albumin, IgM, IgG,PEG-phosphateBlood plasmaU.S. Pat. No. 4,684,723alpha-1-antitrypsin(Tjemeld & Johansson, 1987)Lactate dehydrogenasePEG-aquaphasePig muscleU.S. Pat. No. 6,454,950PPT(Ananthapadmanabhan &Alcaline proteasePEG-sodium sulfateGoddard, 1988)U.S. Pat. No. 4,743,550(Brewer et al., 1988)ProteasePEG-sodium sulfatewhole fermentationU.S. Pat. No. 4,728,613bee(Sieron et al., 1994)Recombinant proteinsPEG-polyvinylalcoholDD 288837(Enfors et al., 1992)Human IgGPEG-phosphateStaphylococcusWO 92/97868(Guiliano & Szlag, 1992)Alcohol dehydrogenasePVP-maltodextrinBaker's yeastU.S. Pat. No. 5,093,254(ADH)(Heinsohne et al., 1992)ChymosinPEG-sodium sulfateAspegillus niger varEP 0 477 284awamori(Kirchberger et al., 1992)Alkalische phosphatasePEG-dextranCalf intestineDD 298424(Dos Reis Coimbra et al., 1994)Beta-lactoglobulinPEG-phosphateCheese whey(Dos Reis Coimbra et al., 1994)Alpha-lactoglobulinPEG-phosphateCheese whey(Cordes & Kula, 1994)Formate dehydrogenasePEG-phosphateCandida biodinii(Hart et al., 1994)IGFPEG-sodium sulfateE. coli(Lorch et al., 1994)EGPEG-ammonium sulfateCellulase mixtureU.S. Pat. No. 5,328,841(Builder et al., 1995)IGF-I or mammalianPEG-citrateE. coliU.S. Pat. No. 5,407,819polypeptide(Heinsohne & Hayenga, 1995)ChymosinPEG sodium sulfateBovine stomachEP 0 477 284(Lee & Khan, 1995)HemoglobinPEG-phosphateBovine bloodU.S. Pat. No. 5,407,579(Braunstein et al., 1995)Different lipases andDetergentsDifferent organismsWO 96/23061proteases(Guinn, 1997)Recombinant hemoglobinPEG-magnesiumE. coliU.S. Pat. No. 5,907,035sulfate(Hayenga et al., 1999)Human growth hormonePEG-ammonium sulfateE. coliU.S. Pat. No. 6,437,101(Tjerneld et al., 2002)BSAHM-EOPO-waterBlood plasmaU.S. Pat. No. 6,454,950(Ageland et al., 2003)Apoliprotein A and EEO sub 30 PO-ReppalE. coliU.S. Pat. No. 6,559,284PES
However, the mechanisms governing the partition of biological materials is still not well understood. It depends on many factors listed in Table 3 below. The most commonly used in current practice are concentration and molecular weight of phase-forming polymers, the type and quantity of the salt and the type and concentration of additives (usually inorganic salts). These factors are generally viewed as the most important to manipulate partitioning of protein to achieve better separation. Therefore, it is extremely difficult to find the appropriate A2PS system for a given protein to be purified from a given source, also because the protein intended for therapeutic use must remain fully functional both in terms of structure (e.g. no aggregation, truncations) and in terms of function.
TABLE 3Factors capable of steering solute partitioningin aqueous two-phase systemsType of phase-forming polymersaMolecular weight of phase-forming polymersaConcentrations of phase-forming polymersaType of additivebConcentration of additivebTemperaturepHPresence of complex-forming additivescStructural Modificationdain aqueous single polymer-salt systems type and concentration of phase-forming salt is the factor equal to those of phase-forming polymer in two-polymer systems.badditive of low molecular weight, such as inorganic salts, urea, etc., with no specific affinity for the solute.caffinity ligands, such as drugs, triazine dyes, organic complexions, fatty acids, etc.dmodification by chemical, enzymatic, etc. treatment resulting in elimination, incorporation, or alteration of topography of solvent-accessible moieties in the solute molecule.
Interleukin-18 binding protein (IL-18BP) is a naturally occurring soluble protein that was initially affinity purified, on an IL-18 column, from urine (Novick et al. 1999). IL-18BP abolishes IL-18 induction of IFN-γ and IL-18 activation of NF-κB in vitro. In addition, IL-18BP inhibits induction of IFNγ in mice injected with LPS.
The IL-18BP gene was localized to the human chromosome 11, and no axon coding for a Tran membrane domain could be found in the 8.3 kb genomic sequence comprising the IL-18BP gene. Four isoforms of IL-18BP generated by alternative mRNA splicing have been identified in humans so far. They were designated IL-18BP a, b, c, and d, all sharing the same N-terminus and differing in the C-terminus (Novick et al 1999). These isoforms vary in their ability to bind IL-18 (Kim et al. 2000). Of the four human IL-18BP (hill-18BP) isoforms, isoforms a and c are known to have a neutralizing capacity for IL-18. The most abundant IL-18BP isoform, isoform a, exhibits a high affinity for IL-18 with a rapid on-rate and a slow off-rate, and a dissociation constant (Kd) of approximately 0.4 nM (Kim et al. 2000). IL-18BP is constitutively expressed in the spleen, and belongs to the immunoglobulin super family. The residues involved in the interaction of IL-18 with IL-18BP have been described through the use of computer modeling (Kim et al. 2000) and based on the interaction between the similar protein IL-1β with the IL-1R type I (Vigers et al. 1997).
IL-18BP is constitutively present in many cells (Puren et al. 1999) and circulates in healthy humans (Urushihara et al. 2000), representing a unique phenomenon in cytokine biology. Due to the high affinity of IL-18BP to IL-18 (Kd=0.4 nM) as well as the high concentration of IL-18BP found in the circulation (20 fold molar excess over IL-18), it has been speculated that most, if not all of the IL-18 molecules in the circulation are bound to IL-18BP. Thus, the circulating IL-18BP that competes with cell surface receptors for IL-18 may act as a natural anti-inflammatory and an immunosuppressive molecule.
IL-18BP has been suggested as a therapeutic protein in a number of diseases and disorders, such as psoriasis, Crohn's Disease, rheumatoid arthritis, psoriatic arthritis, liver injury, sepsis, atherosclerosis, ischemic heart diseases, allergies, etc., see e.g. WO9909063, WO0107480, WO0162285, WO0185201, WO02060479, WO02096456, WO03080104, WO02092008, WO02101049, WO03013577.
The prior art does not describe a purification process of IL-18BP.